ABSTRACT The goal of this Fast-Track proposal is to develop a novel non-cytotoxic, epigenetic therapeutic for life-long disease modification of sickle cell disease (SCD). SCD is a morbid, life-shortening condition caused by mutated hemoglobin (sickle hemoglobin, HbS). The pathophysiology triggered by sickle hemoglobin is interrupted by another hemoglobin that the body usually produces in utero, fetal hemoglobin (HbF). The epigenetic protein DNA methyltransferase (DNMT1) is known to directly silence HbF. DNMT1 is depleted by the generic drug decitabine (Dec). Moreover, because of its chemistry, Dec can deplete DNMT1 without cytotoxicity. Early-phase clinical trials that repositioned Dec for non-cytotoxic DNMT1-depletion in HU- refractory SCD have demonstrated notable efficacy. Unfortunately, marketed Dec has severe pharmacologic limitations, undermining viability for life-long disease modification: (i) it is rapidly destroyed (within minutes) by the enzyme cytidine deaminase (CDA). This is a significant problem because DNMT1-depletion by Dec is S- phase dependent, in other words, exposure-time dependent; (ii) Very high CDA levels in the intestines and liver are a barrier to oral bioavailability, undermining feasibility for life-long disease modification. To solve these related problems, we combined oral Dec with an inhibitor of CDA, tetrahydrouridine (THU). A Phase I clinical trial has demonstrated that THU-Dec solves the fundamental pharmacologic limitations of Dec alone. However, the Phase I trial was conducted `powder-in-bottle': patients attended the clinic for API reconstituted in the pharmacy then drunk orally. Also, THU, a new chemical entity, is synthesized by a batched, poor-yield, very expensive synthesis scheme (~$70,000/kg). We will address these two major, but technically tractable, hurdles to clinical adoption via the following aims: Phase 1 - Aim 1: Complete process optimization of THU synthesis. Instead of combining hydrogenation and reduction of uridine in a 1-step process, we pursue a 2- step reaction scheme that separately optimizes hydrogenation and reduction reactions, to produce higher, scalable, stereo-selective THU yields at substantially lower costs. We have very promising preliminary data. Phase 2 - Aim 2: Formulate oral THU and oral Dec into a combination drug product that releases THU immediately and Dec about 60 minutes later. We will pursue a combination THU-Dec `tablet-in-tablet' strategy such that THU is released immediately in the stomach but the Dec component, coated with a pH- sensitive polymer, is released in the small intestine (neutral-pH). In vivo studies in a baboon model will be used to confirm that the product recapitulates or improves over `powder-in-bottle' PK. Phase 2 - Aim 3: Conduct a Phase IIa clinical study to confirm the dose of the combination drug product to be used in subsequent studies. The body of scientific, safety and efficacy data already generated for THU and Dec has de-risked this endeavour to an extent very unusual for most early stage drug development efforts; completion of this project promises rapid movement through pivotal clinical trials, to NDA, and to patients in need.